tbuckley/cabot-search huggingface dataset:
Abstract P1182: The Genetic Dissection Of <i>Rpl3l</i> As A Causal Gene For Neonatal Dilated Cardiomyopathy And Left Ventricular Noncompaction
Authors: Buyan-Ochir Orgil, Qingqing Gu, Hugo Martinez, Akhilesh Bajpai, Mohammed Absi, Kaitlin Ryan, Neely Alberson, Lu Lu, Jeffrey A Towbin, Enkhsaikhan Purevjav
Journal: Circulation Research (2023)

Background: Cardiomyopathies are responsible for nearly 10% of cardiac deaths in neonates. Recently, biallelic variants in ribosomal protein L3-like ( RPL3L , NM_005061.2) have been reported in neonates with dilated cardiomyopathy (DCM) and heart failure (HF). This study is aimed to study RPL3L induced genetic pathways and mechanisms involved in the development of DCM. Methods: Whole exome and RNA sequencing, histology, immunohistochemistry, and Western blotting were performed in a human explanted heart. Genetic correlation, expression quantitative trait loci (eQTL) mapping and functional analysis were performed using cardiac gene expression data from the patient as well as human and murine genetic reference populations (GRPs). Results: Two-month-old patient with DCM and HF carrying compound heterozygous RPL3L variants, p.A51T and p.V231F, has been managed on an INTERMACS profile 2 with continuous-flow ventricular assist device and successfully bridged to heart transplantation at the age of 4 months. Morphology of the explanted heart displayed apical deep intertrabecular recesses consistent with left ventricular noncompaction (LVNC). Histology revealed interstitial fibrosis and myocardial hypertrophy. No signs of myocarditis and glycogen or mucopolysaccharides deposits were detected. Immunohistochemistry revealed abnormal clusters of RPL3L protein in the cytosol of cardiomyocytes and disruption of F-actin, Z-disk proteins, MYL4 and dystrophin was evident. In a mouse heart, the Rpl3l level was of 33.45 RPKM, while levels of RPL3L appeared to be 32 RPKM in a human heart compared to its mean expression in other adult tissues of 2.54 (mouse) and 2.36 (human). Systems genetics analysis identified high expression values ranged from 11.31 to 12.16 across murine GRPs of BXD mice with the ~1.8-fold difference. Pathways such as “thermogenesis”, “diabetic cardiomyopathy” and “DCM” significantly associated with RPL3L. e QTL mapping suggested Myl4 (Chr 11) and Sdha (Chr 13) as the upstream regulators of Rpl3l . Conclusions: Compound RPL3L heterozygosity is causal for neonatal DCM and LVNC. The RPL3L is highly expressed in the heart tissue of humans and mice. Genes, Myl4 and Sdha, are strong candidates that regulate expression of Rpl3l in murine heart.
Abstract GS.103: Human Cardiomyopathy Disease Variant In Formin Homology 2 Domain Containing 3 (FHOD3) Protein Revealed Novel Early Autophagy Dysregulation And Mitochondrial Dysfunction
Authors: Asma Boukhalfa, Sarah N Powers, Gregory Martin, Robert M Blanton, Gordon S Huggins, Howard H Chen
Journal: Circulation Research (2023)

Introduction: The FHOD3-V1151I variant has been identified by multiple Genome Wide Association Studies to be associated with hypertrophic cardiomyopathy (HCM). FHOD3 regulates actin filament formation which is central to key cardiomyocyte processes including autophagy. The precise role of autophagy in cardiomyopathy is an emerging area of research but poorly characterized. We hypothesize that V1151I may perturb FHOD3-dependent cardiac autophagy regulation. Methods: The homologous V1151I variant was introduced into mice using CRISPR. Cardiac function was studied by echocardiography, cardiac mitochondrial function measured by Seahorse (Agilent) and oil red O staining of lipid droplets in cardiac sections. Cultured H9C2 cardiomyoblasts were transfected with FHOD3-V1151V (WT) or V1151I (variant) expression plasmids and immunoprecipitation and immunofluorescence were performed to assess protein interaction and localization. Phosphatidylinositol 3-phosphate (PI3P, Echelon) was used for the rescue experiments. Results: Heart tissue from FHOD3-V1151I variant mice (n=7) showed a 5-fold (p&lt;0.0001) increase in lipid droplet accumulation and impaired mitochondrial respiration and ATP production (p&lt;0.0001), while cardiac structure and function assessed by echocardiography was similar to V1151V WT littermate control hearts (n=7). A bioinformatics analysis predicted FHOD3 interaction with PFN4 which we confirmed by co-immunoprecipitation in addition to ATG16, a regulator of autophagic vesicle membrane biogenesis. The FHOD3-PFN4-ATG16 interaction was however lost in FHOD3-V1151I hearts. Immunofluorescence of H9C2 cells expressing FHOD3-V1151I revealed that the variant disrupts interaction of LC3 with PI3P, a hallmark of early autophagy. The disrupted LC3-PI3P interaction seen with the V1151I variant phenocopies cells treated with early autophagy inhibitors 3-MA or spautin, but not late autophagy inhibitor chloroquine. Furthermore, exogenous PI3P restored the molecular interaction and autophagy dysregulation. Conclusions: We demonstrate for the first time that early autophagy dysregulation underlies energetic defects observed in cardiomyopathy, and is reversible, thus providing a novel potential therapeutic target.
Abstract P3156: Activated Fibroblast-Derived Exosomal Mir-216a-5p Promotes Endothelial Dysfunction By Targeting PGM5 In Diabetic Heart
Authors: Prabhat Ranjan, Roshan Dutta, Karen Colin, Yajing Wang, Suresh K Verma
Journal: Circulation Research (2023)

Background: Diabetic patients are more susceptible to heart failure (HF). High blood glucose level in diabetic patients eventually triggers the body's inflammatory response and causes cardiac fibroblast activation, endothelial cell dysfunction (ECD), and, ultimately, HF. Previously, we have shown that activated fibroblast-mediated ECD leads to HF. However, the molecular mechanism of fibroblast-induced ECD in diabetics is not yet well defined. Therefore, we hypothesized that “myofibroblast in the diabetic heart secretes exosomes loaded with antiangiogenic/profibrotic factors, which impair EC function.” Methods: Exosomes were isolated from diabetic mice plasma and fibroblast conditioned media by ultracentrifugation and characterized by nanosight &amp; electron microscopy. We cultured mouse primary heart endothelial cells in EC growth media. ECs were treated with exosomes derived from fibroblasts (treated with either 25mM glucose, 500nM Angiotensin II (AngII) or both) for 48 hr. Mannitol (25mM) served as control. Pathway-based miRNA array was screened with exosome-derived miR, and targets were predicted bioinformatically for selected miR. Results: Glu-AngII cotreatment significantly activated fibroblasts as shown by qPCR (Col1α, and FN expression) and western blot (pSmad2/Smad2, p-p38/p38). Interestingly, exosomes derived from activated fibroblasts significantly induced ECD (eNOS, VEGF, CD31 genes, and proteins expression). We further checked the effect of diabetic mice plasma exosomes on ECs function. We found significantly impaired endothelial function, as shown by Matrigel tube formation and Boyden chamber migration assays. Micro RNA array and qPCR data showed that miR-216a-5p was highly upregulated in exosomes derived from FBs cotreated with Glu-AngII. Pathway-based analysis revealed that PGM5 could be a potential target in miR-216-induced ECD. Interestingly, miR-216a-5p inhibition significantly rescued diabetes-induced PGM5 inhibition and improved endothelial function Conclusions: Taken together, this study demonstrates that fibroblast in the diabetic heart releases miR-216a-5p through exosomes, which promote endothelial dysfunction via the miR-216a-5p/PGM5 axis.
Abstract P3080: Macrophages Synthesize Bioactive Lipids To Modulate Cardiomyocyte Metabolism And Proliferation During Cardiac Regeneration
Authors: Connor Lantz, Amanda C Becker, Mallory Filipp, Edward B Thorp
Journal: Circulation Research (2023)

Introduction: Once thought to be a quiescent process, elimination of dying cells by macrophages actively reprograms macrophage metabolism to support downstream functions. Previously, we discovered that the phagocytic receptor MerTK is necessary for regeneration in the neonatal murine heart. Single-cell RNA sequencing and bulk-tissue lipidomics revealed prioritization of arachidonic acid metabolism towards the synthesis of Thromboxane A 2 (TXA 2 ) in regenerating hearts, yet the molecular mechanisms directly connecting macrophage-derived TXA 2 to regeneration remained undefined. Therefore, we sought to determine if TXA 2 and activation of its receptor ( Tbxa2r ) on cardiomyocytes is necessary for cardiac regeneration. Results: We created a novel mouse to conditionally knockout Tbxa2r in cardiomyocytes using the CRE recombinase under the Myh6 promoter. Loss of Tbxa2r did not affect normal cardiac development but significantly impaired cardiac regeneration as seen by significant fibrotic scarring, reduced cardiac output, and decreased cardiomyocyte proliferation. We next asked how Tbxa2r initiates intracellular signaling to support cardiomyocyte proliferation. Knowing that the YAP/TAZ pathway has been previously described to induce cardiomyocyte proliferation during regeneration and is regulated by GPCRs, we found that TXA 2 mimetics activate TBXA2R leading to the stabilization of YAP/TAZ in a time- and dose-dependent manner. Furthermore, YAP was translocated into the nucleus of cardiomyocytes in the border zone 3 days post-MI. However, deletion of Tbxa2r in cardiomyocytes completely prevents YAP translocation into the nucleus. Lastly, we have found that a TXA 2 mimetic increases the glycolytic capacity of cardiomyocytes further supporting anabolic processes needed for proliferation. Excitingly, we also observed an increase in glycolytic enzyme transcripts at the border zone of the infarct using publicly available spatial transcriptomic data. Conclusions: Our findings advance our understanding of how cell death, removal and recycling regulate macrophage metabolism, resulting in the production of bioactive lipids that further regulate the metabolism of neighboring cardiomyocytes during regeneration.
Abstract P3157: MicroRNA-16 Exerts Anti-fibrotic Effects Via Suppression Of Tgfbr3 In Rats With Heart Failure And Preserved Ejection Fraction
Authors: Jae Hyung Cho, Alex Rajewski, Catherine Bresee, Lizbeth Sanchez, Myung Yoon Kim, Thassio Mesquita, Xaviar Jones, Isabelle Pellot, Weixin Liu, Asma Nawaz, Ahmed G Ibrahim, Russell Rogers, Eugenio Cingolani, Eduardo Marban
Journal: Circulation Research (2023)

Background: Cardiosphere-derived cells (CDCs) reverse diastolic dysfunction by attenuating ventricular fibrosis in rats with heart failure and preserved ejection fraction (HFpEF). MicroRNAs packaged in exosomes secreted from CDCs (CDCexo), are discovered to mediate the beneficial effects of CDCs. Hypothesis: The anti-fibrotic effects of CDCs are mediated by microRNAs contained in CDCexo. Methods: Dahl salt-sensitive rats were fed either a high-salt diet to induce HFpEF, or a normal-salt diet to serve as controls. Echocardiogram-verified HFpEF rats were randomized to receive human CDCexo (n=37) vs. vehicle (n=47) via tail vein weekly for 4 weeks. Control rats (n=16) served as the normal phenotype. RNA sequencing of CDCexo was performed to search for candidate microRNAs. Single-cell RNA sequencing was performed in isolated ventricular cells to investigate the mechanisms of CDCexo. Five most abundant microRNAs were selected and injected via tail vein to HFpEF rats. MicroRNA-injected HFpEF rat hearts underwent single-cell RNA sequencing to investigate its mechanisms. Results: HFpEF rats injected with CDCexo demonstrated improved diastolic function compared to HFpEF rats injected with vehicle (E/E’ ratio 12.1±2.6 vs. 18.7±4.6 in vehicle, p&lt;0.0001). Masson’s trichrome staining revealed reduced ventricular fibrosis in CDCexo rats (3.5±1.4 vs. 5.4±1.4 % in vehicle rats, p=0.016). Negative regulation of transforming growth factor beta (TGF-β) receptor signaling pathway (p=0.002) was identified in isolated cardiac fibroblasts after CDCexo injection. MicroRNA-16 was revealed as the most abundant microRNA in CDCexo, and intravenous delivery of microRNA-16 recapitulated the beneficial responses (improvement of diastolic function and attenuation of ventricular fibrosis) of CDCexo in rats with HFpEF. Single-cell RNA sequencing of microRNA-16-injected HFpEF hearts confirmed down-regulation of TGF-β receptor activity (p=0.018) in cardiac fibroblasts via suppression of Tgfbr3. Conclusion: CDCexo improved diastolic function by suppressing ventricular fibrosis in rats with HFpEF. MicroRNA-16, the most abundant microRNA in CDCexo, recapitulated the beneficial responses of CDCexo in HFpEF rats, via suppression of Tgfbr3 in cardiac fibroblasts.
Abstract P3194: Redd1 Is Required For Glucose Metabolism And Insulin Sensitivity In Cardiomyocytes
Authors: Mason Wheeler, Shaunaci Stevens, Jamie Renick, Michael Dennis, Jessica M Pfleger
Journal: Circulation Research (2023)

Type II diabetes (TIID) is characterized by insulin resistance (IR) and doubles a patient’s risk for cardiovascular disease (CVD). The pathogenesis of CVD in patients with TIID, or diabetic cardiomyopathy (DC), is unclear. Regulated in development and DNA damage (REDD)1 is a negative regulator of mammalian target of rapamycin complex (mTORC)1. Notably, deletion of REDD1 leads to systemic IR in mice. REDD1 is, therefore, necessary for insulin sensitivity, however the mechanisms required remain unclear. We hypothesized that REDD1 is required for glucose oxidation and, therefore, insulin sensitivity in cardiomyocytes. To examine the role of REDD1 in cardiomyocyte glucose metabolism and energetics, we cultured wild type (WT) AC16 cardiomyocytes or those with REDD1 deletion with 5.5 mM glucose and examined cellular respiration. The data demonstrate compromised respiration, as indicated by reduced maximal respiratory capacity in REDD1-null versus WT cells (1.5-fold±0.214). Importantly, western blotting confirmed the absence of REDD1 in cells with REDD1 deletion, as well as increased phosphorylation of eukaryotic translation initiation factor 4E-binding protein (p4E-BP)1 (Thr37/46) (1.3-fold±0.085), a direct target of mTORC1. Thus, REDD1 inhibits mTORC1 signaling and enhances glucose-driven respiration in cardiomyocytes. To examine the mechanisms by which REDD1 supports glucose utilization, we examined levels of phosphorylated pyruvate dehydrogenase (pPDH) (Ser293). Our data show that pPDH is increased in REDD1-null cells versus WT controls (1.2-fold±0.054), as well as in hearts from REDD1-deficient mice (1.2-fold±0.013), suggesting reduced glucose oxidation. We also observed enhanced extracellular acidification, an indirect measure of glycolysis, in our REDD1-null cells versus WT controls (1.7-fold±0.136). Pyruvate dehydrogenase kinases (PDKs) phosphorylate PDH. In our REDD1-null cells, we observe a significant increase in PDK4 mRNA versus WT controls (8-fold±0.593), as assessed by qPCR and consistent with the diabetic heart. Together, these data suggest that REDD1 inhibits mTORC1 activity and the expression of PDK4, activating PDH, and glucose oxidation as the mechanism by which cardiomyocytes remain insulin sensitive.
Abstract P2099: Small Proline-rich Protein 1a Is A Key Downstream Effector Of Microrna-150 During Both Maladaptive Cardiac Remodeling In Mice And Human Cardiac Fibroblast Activation
Authors: Marisa Sepúlveda, Taiki Hayasaka, Il-man Kim
Journal: Circulation Research (2023)

Background &amp; Objectives: MicroRNA-150 (miR-150) is conserved between rodents and humans, is significantly downregulated during heart failure (HF), and correlates with patient outcomes. We reported that miR-150 is protective during myocardial infarction (MI) in part by decreasing cardiomyocyte (CM) apoptosis and that proapoptotic small proline-rich protein 1a ( Sprr1a ) is a direct CM target of miR-150. We also showed that Sprr1a knockdown in mice improves cardiac dysfunction and fibrosis post-MI and that Sprr1a is upregulated in pathological mouse cardiac fibroblasts (CFs) from ischemic myocardium. However, the direct functional relationship between miR-150 and SPRR1A during both post-MI remodeling in mice and human CF (HCF) activation was not established. Methods &amp; Results: Here, using a novel miR-150 knockout (KO); Sprr1a -hypomorphic ( Sprr1a hypo/hypo ) mouse model, we demonstrate that Sprr1a knockdown blunts adverse post-MI effects caused by miR-150 loss. MiR-150 KO; Sprr1a hypo/hypo mice also exhibit decreased profibrotic markers in the infarct areas of hearts as compared to miR-150 KO mice. Moreover, primary adult HCF studies reveal that SPRR1A is upregulated in hypoxia/reoxygenation-treated HCFs and is downregulated in HCFs exposed to the cardioprotective β-blocker carvedilol, which is inversely associated with miR-150 expression. Significantly, we show that the protective roles of miR-150 in HCFs are directly mediated by functional repression of profibrotic SPRR1A (See Figure). Conclusions: These findings delineate a pivotal functional interaction between miR-150 and SPRR1A as a novel regulatory mechanism pertinent to CF activation and ischemic HF.
Abstract P3126: Atrial Fibrillation Is Associated With Decreased Claudin-5 In Cardiomyocyte
Authors: Tao Luo, Baihe Chen, Haiqiong Liu, Yuanzhou Wu, Ahmed Abdel-Latif, Jin Kuyng Kim, Masafumi Kitakaze
Journal: Circulation Research (2023)

Background: Although it is critically important to understand the underlying molecular and electrophysiological changes that predispose to the induction and maintenance of atrial fibrillation (Af), the underlying mechanism of Af is still poorly defined. Af is characterized as the electrophysiological and membrane integrity abnormality of the atrial cells, and claudin-5, a tight junction protein, may be involved in the pathophysiology of Af, however, the role of claudin-5 in Af is unknown. Methods and Results: Cardiac tissues from the left atrial appendage were obtained from AF patients with rheumatic valvular heart disease undergoing modified radiofrequency ablation maze procedure with approval from local human research ethic review and normal left atrial appendages were obtained from non-AF donors. Western blot, immunofluorescence and transmission electron microscope showed the expression of claudin-5 significantly decreased in cardiomyocytes from left atrium of AF patients compared to non-AF donors. Then proteomics analysis was performed to screen the specific protein expression and signal pathway changed in AF heart tissue vs. non-AF heart tissue, and showed that 83 proteins were downregulated and 102 proteins were upregulated in the left atrial appendage of AF patients. Among them, DAG, CACNA2D2, MYL2 were downregulated and MCU was upregulated. KEGG pathway analysis showed these changes would lead to hypertrophic and dilated cardiomyopathy. Finally, claudin-5 shRNA AAV was injected in mouse left ventricle to knockdown claudin-5 in cardiomyocytes to observe whether change of claudin-5 affects the protein expression in KEGG pathway analysis. We found claudin-5 deficiency caused severe ventricular dilation, ST elevation and arrythmia, and the protein level of DAG, CACNA2D2 and MYL2 were downregulated and MCU was upregulated. Co-immunoprecipitation also confirmed the specific protein-protein interaction between these targeted proteins and claudin-5. Conclusion: We demonstrated that the decrease of claudin-5 in the left atrium of AF patients with rheumatic valvular heart disease. The mechanism of AF might be associated with claudin-5 downregulation-induced decrease of DAG, CACNA2D2, MYL2 and increase of MCU in cardiomyocytes.
Abstract P1170: Changes In Endothelin Signaling In A Long-term Rat Coronary Culture Resembles Chronic Coronary Syndrome
Authors: Spyridoula Kazantzi, Lars Edvinsson, Kristian A Haanes
Journal: Circulation Research (2023)

Background: Coronary artery disease generates changes in the structure and function of the blood vessels. 48hrs organ culture (mimicking the acute loss of blood flow) of coronary arteries induces the upregulation of contractile endothelin type B (ETB) receptors, resembling the pattern seen in ischemic heart disease. MEK inhibitors (U0126 and Trametinib), can acutely inhibit the upregulation of these receptors, illustrating the importance of this pathway. The 48hrs organ culture model has been proven successful for translation in acute ischemic disease, but arterial changes have not been studied in a long-term chronic model which could be important for chronic coronary syndrome. Purpose: 1) Investigate the changes of ETB receptors after incubation in a long-term optimized medium for organ culture, mimicking the reduction of blood flow in chronic coronary syndrome and 2) determine whether Trametinib, would have treatment effect on the upregulation of ETB receptors in both long term and short term culture. Methods: Rat coronary arteries were incubated in organ culture for 48hrs in DMEM ± trametinib (“acute”) or in a long-term optimized medium for organ culture for 14 days (“chronic”). After the incubation, arteries were mounted stainless steel wires (40 μm) on a Mulvany-Halpern arterial wire myograph and cumulative concentration response curves were made to measure arterial contractility to the ETB receptor specific agonist Sarafotoxin (S6c). Trametinib was added at time 0 (for both the acute and chronic organ culture) or 7 days delayed. Results: For the “acute” culture, we observed a high contractile effect in response to S6c (Emax=136.5±18%). The presence of Trametinib greatly reduced this contractile effect (Emax=10±1.9%) and arteries were comparable to fresh (Emax=0.8±0.7%). The rat coronary arteries in the “chronic” culture maintained their ability to contract in response to potassium, and S6c had a strong contractile effect (Emax=74.64±17%). Interestingly, neither trametinib addition at time 0 nor at day 7 could inhibit the functional upregulation of ETB in the chronic model. Conclusion: Chronic coronary syndrome may require new inhibitors of ETB receptor upregulation. This could be investigated with our new long-term rat coronary artery model.